A cell-free system for DNA replication has been reconstituted from partially purified enzymes isolated from unfertilized eggs of the frog, Xenopus laevis. This system has been shown to initiate DNA replication in isolated nuclei, with purified DNA molecules, or with purified polyoma mini-chromosomes. During the course of this project each of the components involved in initiation of DNA replication will be identified, purified to electrophoretic homogeneity, and characterized. Cytoplasmic components currently being purified are initiation factor (I factor), DNA polymerases X-I, X-II, and X-III, DNA polymerase II, relaxation protein, unwinding protein, DNA ligases I and II, and ribonuclease H. In addition, the time of synthesis and cytoplasmic localization of each of the components is being examined. In order to improve the electron microscope assay for the initiation of DNA replication in vitro, a series of well defined internal standard DNA molecules and single enzyme reaction products is being characterized. Finally, a model for DNA replication is being developed in which both positive control elements (I factor) and negative control elements (I repressor, DNA polymerase inhibitor) are coordinated to regulate DNA replication during early embryogenesis in Xenopus laevis. BIBLIOGRAPHIC REFERENCES: Benbow, R.M., Joenje, H., White, S.H., Breaux, C.B., Krauss, M.R., Ford, C.C., and Laskey, R.A. "Cytoplasmic Control of Nuclear DNA Replication in Xenopus laevis" in International Cell Biology 1976-1977. In Press, The Rockfeller University Press, New York, 1976. Benbow, R.M. and Laskey, R.A. "Replication of Polyoma DNA Molecules Microinjected into Xenopus Laevis Eggs", J. Mol. Biol., submitted, 1976.